1. Field of the Invention
The present invention relates to methods and compositions useful in cancer therapy. A preferred embodiment relates to the use of antisense oligonucleotides to purge tumor cells from bone marrow prior to autologous bone marrow transplantation. With this novel treatment, the bone marrow cells are obtained from an individual, and exposed to a proliferation-inhibiting amount of an oligonucleotide having a sequence complementary to a sequence of RNA transcribed from a gene present in the cancerous cells. The cells are then infused back into the individual. This treatment preferentially inhibits the proliferation or kills malignant cells but not normal cells. Preferably, the gene encodes protein P53, although other antisense oligonucleotides may also be used. The invention also includes chemotherapeutic procedures and compositions for in vivo administration.
2. DESCRIPTION OF THE RELATED ART
This discussion illustrates the problems faced by clinical investigators seeking to improve cancer treatments. A number of references have been included for the convenience of the reader. Inclusion of these references is not an admission that such references represent prior art with respect to the present invention.
Cancer is one of the most feared diseases of our age, causing many thousands of deaths each year. Unfortunately, despite substantial advances in cancer therapy, significant room for improvement remains. Many of the antineoplastic agents currently used in cancer treatment have an unacceptably low therapeutic index; in other words, these agents are unable to block proliferation of or kill malignant cells at doses that do not cause unacceptable toxicity in normal tissue. Consequently, treatment with these agents causes undesirable side effects, and in many circumstances, an effective dose of these agents may even be fatal to the host. Therefore, for many years, cancer researchers have sought to develop a method for treating cancer that would allow one to selectively kill the cancer cells without destroying normal host tissue.
Autologous bone marrow transplantation (ABMT) represents a significant advance in this area. With this technique, one first removes bone marrow cells from an individual having cancer, and then extensively treats the patient with radiation, antineoplastic agents, or a combination of both at a dose sufficient to eliminate most, if not all, of the malignant cells. Unfortunately, this treatment often kills normal hemopoietic cells as well. As a result, it is necessary to reconstitute the patient's hemopoietic system by transplanting the marrow removed prior to treatment. This procedure has been described by Chang, et al. Lancet, pp. 294-295 (Feb. 8, 1986), and in a monograph entitled Autologous Bone Transplantation: Proceedings of the Third International Symposium, Dicke, et al., (eds), The University of Texas M. D. Anderson Hospital and Tumor Institute at Houston (1987), both of which are expressly incorporated herein by reference.
One major disadvantage associated with ABMT is the fact that cells of the bone marrow graft are often contaminated with malignant cells. This is particularly true when the patient suffers from a malignancy of the type known to invade or originate from bone marrow, for example, leukemias, lymphomas, myeloma, breast cancer, and small cell carcinoma of the lung.
Existing attempts to reduce or eliminate malignant cells from bone marrow involve the use of either monoclonal antibodies, drugs, or simple in vitro culture. However, these existing purging methods have well documented inherent shortcomings. For example, techniques that employ antibodies are dependent on the antigen density of the tumor cells, which may, in some cases, be quite low (Boyle and Gee, Cancer Investigation 5:113-118 (1987)). In addition, heterogeneity of tumor antigens complicates selection of a monoclonal antibody that is specific for a given tumor. In one study, mafosfamide has been reported to be more effective than monoclonal antibodies for bone marrow purging prior to transplantation. However, despite this report that this procedure improved clinical outcome in patients with acute leukemia, the procedure was not curative. (Gorin, Exp. Hemat. 16:415 abstract 9, 1988). Other investigators report that there is a significant progressive shift from malignant to normal myelopoiesis when marrow from donors with acute or chronic myelocytic leukemia is maintained in culture up to about 4 weeks. This finding has led to attempts to purge marrow by in vitro culture (Coulumbel, et al., J. Clin. Invest. 75:961, 1985; Eaves, et al., Haem. Blood Transfusion 29:163, 1985) prior to autologous bone marrow transplantation. Although one group reports that this procedure appeared to cure one patient with acute myelogenous leukemia (Chang, et al., Lancet 1:2914, 1986), in general, the continuing presence of malignant hemopoiesis throughout the culture period limits the therapeutic value of this technique.
Recently, it has been learned that expression of certain genes involved in cellular proliferation can be inhibited by treating cells expressing those genes with "antisense" molecules. These antisense molecules have a sequence that is complementary to a portion of the RNA transcribed from the selected gene. Although the exact molecular mechanism of inhibition is not known, it has been suggested to result from formation of duplexes between the antisense molecule and RNA transcribed from the target gene. The duplexes may inhibit translation, processing, or transport of mRNA sequence or may lead to digestion by the enzyme RNaseH.
Very recent studies involving the use of antisense oligonucleotides have been reviewed by Stein and Cohen, Cancer Res. 48:2659 (1988). Several types of antisense molecules have been screened for their ability to inhibit the synthesis of particular proteins using both intact cells and in vitro systems for protein synthesis. See Paoletti, Anti-Cancer Drug Design 2:325 (1988). For example, agents with specificity for RNA transcribed from the myc gene have been reported to inhibit the proliferation of the human AML line HL60 (Wickstrom, et al., Proc. Natl. Acad. Sci. USA 85:1028 (1988) and normal T lymphocytes (Heikkila, et al., Nature 328:445 (1987), and oligodeoxynucleotides complementary to cyclin mRNA have been reported to suppress the division of 3T3 cells. (Jaskulski, et al. 1988). In addition, in a murine system, transfection of a plasmid encoding antisense p53 RNA into transformed MethA or non-tranformed NIH3T3 fibroblasts was reported to reduce the growth rate of the transfectants. Of course, studies in the mouse may have very limited applicability to human systems, particularly where, as with p53, significant differences exist between the structure of the murine gene and the human gene given the same designation. Lamb and Crawford Mol. Cell Biol. 6:1379 (1986).
Unfortunately, although many of the references cited above are of general scientific interest, they suffer from a number of shortcomings that render them insufficient to establish efficacy of oligonucleotides as chemotherapeutic agents for cancer. For example, none of the studies reported to date indicate that treatment with selected antisense oligonucleotides would be likely to have an acceptable therapeutic index. It has not been shown that antisense oligonucleotides are lethal for or even inhibitory for proliferation of malignant cells but not corresponding normal cells under the same treatment conditions. In addition, these studies do not describe the effects of antisense oligonucleotides on fresh tumor cells or fresh normal cells; instead, the studies utilized continuous cell lines which differ in many respects from fresh cells and whose relevance as a model for screening of chemotherapeutic agents has been repeatedly challenged by members of the scientific community. Vinditti, Seminars in Oncology Vol. 8, p. 349 (1981). Fortunately, with the present invention, these shortcomings have been overcome and the therapeutic potential of chemotherapy with selected antisense oligonucleotides convincingly established for the first time.
Moreover, the present inventor has now discovered that certain antisense oligonucleotides, and in particular, an oligonucleotide complementary to the mRNA encoding the human p53 gene product, may be successfully used to selectively eliminate malignant cells from bone marrow prior to autologous bone marrow transplantation. These and other advantages of the invention are described more fully below.